The overall goal is the understand mechanisms underlying arrhythmia formation and cellular injury at boundaries between ischemic and non- ischemic myocardium, that is the ischemic "border zone". Our hypothesis is that diffusion of gases, substrates, metabolites and endothelial derived factors across these boundaries are responsible for the unique ionic and electrophysiologic characteristics of the border zone. Previous work by us and others suggest that modulation of pHi and pHo by CO2 may have direct effects on cellular K+ loss, lactate production, depolarizing and repolarizing current and SR Ca2+ release. Taken together these results indicate that CO2 diffusion and accumulation in the border zone might contribute directly to the mechanisms responsible for the occurrence of arrhythmias and cellular injury. We plan to focus on cytosolic pHi and its regulation in the border zone and relate these changes to Ca2+-dependent processes that include impulse propagation, spontaneous membrane depolarization, propagated calcium waves, cell-to-cell electrical coupling and cellular injury. These Ca2+-dependent effects are predicted to be dependent on H+ production and transsarcolemmal flux. As such, C02 diffusion, washout of the extracellular space and the energy demands are predicted to influence these Ca2+-dependent effects. The specific aims are: (1) to evaluate the role of CO2 and O2 diffusion, and transsarcolemmal H+ flux for cellular Ca2+ loading in the border zone. (2) to determine if the ischemic subendocardial cell layers are more susceptible to cellular uncoupling, discontinuous impulse propagation and cellular injury compared to intramural layers due to greater cellular Ca2+ loading. (3) to evaluate mechanisms for the initiation of premature ventricular beats in the border zone, namely the pH-dependence of the formation and propagation of Ca2+ waves. (4) to determine the role of vasoactive peptides and endogenous endothelial derived factors for the modulation of pHi and Ca2+i in the border zone. The specific aims will be studies with two models of ischemic boundaries. Confocal fluorescent videomicroscopy of parameter specific fluoroprobes, and passive recording electrode assays will be used to characterize the influence of CO2 and O2 on the mechanisms of pHi and Ca2+i regulation within the border zone simulated in a monolayer of cultured neonatal cardiac monocytes. In other experiments confocal fluorescent videomicroscopy, sillicon/iridium microelectrodes and histological methods will be used the isolated arterially perfused and ischemic papillary muscle to determine the relationship between the rise of Ca2+i, cellular uncoupling, impulse propagation and cellular injury.